Chemotaxis, the ability to sense chemical gradients and alter motility accordingly, enables bacteria to engage in complex behaviors, including biofilm formation and colonization of a variety of hosts. Human colonization by some bacteria can cause serious diseases, while colonization by others is actually beneficial. Understanding the mechanisms bacteria use to sense and respond to chemotactic stimuli should foster development of new therapeutic strategies for treating and preventing bacterial infections, as well as potentially avoiding negative side effects on beneficial microflora. The long-term goal of this project is to elucidate the cytoplasmic signaling mechanism of the E. coli serine chemoreceptor, Tsr. This is a well-established model system for studying bacterial chemotaxis and will allow for in vivo investigation of the Tsr signal transduction mechanism. Tsr is a transmembrane receptor containing a periplasmic ligand-binding domain and a cytoplasmic portion composed of HAMP, sensory adaptation or methylation, and signaling helix bundles. Serine binding promotes Tsr conformational changes that traverse the cytoplasmic membrane and impinge on the HAMP domain, which in turn modulates receptor output signals by controlling activity of the Tsr-bound kinase CheA. The overall objectives of this proposal are to identify structural elements in the methylation helix (MH) bundle that produce different output states of the receptor, to elucidate the signaling role of highly conserved sequence features in the MH bundle, and to investigate the structural interplay between HAMP and the MH bundle. Recent studies suggest that signal transmission in transmembrane receptors may occur through a dynamic interplay between neighboring subdomains coupled in structural opposition. This project will test that working hypothesis by altering conserved structural motifs in the HAMP and MH bundles of Tsr and characterizing the signaling properties of those mutant receptors with in vivo behavioral assays and in vitro structural studies.